Specific targeting agents bearing a detectable moiety offer the potential for earlier diagnosis of disease, if an increased amount of the targeting-detectable conjugate is localized to a greater extent in tissue to be imaged compared to background tissues. In practice, detectable agent in background tissue needs to be minimized while detectable agent in target tissue needs to be maximized. Radioactive nuclides are preferred detectable agents and nuclides such as Tc-99m and In-111 are widely used for scintigraphy and single photon emission computed tomography. However, there are limitations on the sensitivity of these detection modalities. For instance, even for specific targeting of tumor tissue in a patient, tumor nodules of less than 1 centimeter diameter may be very difficult to detect. A superior imaging modality is offered by positron emission tomography (PET), which offers the promise of a dramatic increase in sensitivity, and therefore the ability to detect disease at an earlier stage. What has mainly hampered development of PET into a routinely applied clinical diagnostic modality are the inadequate techniques related to radiolabeling of specific targeting agents with useful PET nuclides.
There are about 20 nuclides of theoretical utility for PET in that they have positron decay and a suitable half-life (minutes or longer). In practice, most of these nuclides are really unsuitable for a variety of reasons, including, several separate reasons in the case of many of them. These reasons include but are not limited to, availability and cost of parent nuclides, nuclide preparation issues related to target preparation and bombardment, handling and shipment of the nuclide, cyclotron size and energy etc., chemical separation issues, radiolabeling issues including on-site radiolabeling issues, and decay energy and properties of the PET nuclides themselves which often include other beta and/or gamma decay. These extensive and severe practical matters can essentially preclude most nuclides from consideration as viable PET agents, and the two most commonly considered nuclides for PET, fluorine-18 and gallium-68, retain the best combination of features, and fewest problems among PET nuclides. Gallium-68 (Ga-68) has two great advantages over F-18 when considering both nuclides for PET usage. First, it is available from a generator, which makes it available on site by a simple ‘milking’ process that can be carried out daily, or even hourly. This makes Ga-68 independent of the need for a nearby cyclotron, as is needed for F-18. Second, it is a radiometal and can be complexed by suitable chelating agents. In contradistinction, fluorine is mainly available as F-18 fluoride ion in aqueous solution and this must be taken into a dry organic environment prior to chemical manipulations to covalently attach it to targeting agents of choice. The half-lives of both nuclides are short (F-18˜2 h; Ga-68˜68 minutes) and the intense positron energy emitted makes chemical manipulations far from trivial and possibly extremely hazardous to technical personnel. Given the chemistry needed for F-18 attachment to targeting agents, the radiochemical processes can only be carried out in custom-designed dedicated facilities, and these facilities must also be located near a cyclotron that produces the F-18 raw material.
Gallium-68 does not suffer from these drawbacks. It is available from a long-lived parent nuclide (germanium-68; half-life 288 days) that can be adsorbed to various solid phases, from which the Ga-68 can then be selectively eluted. Thus, a Ga-68 generator can be fabricated, and several have been described (Ambe, 1988; Greene and Tucker, 1961; Loc'h, 1980; Lewis 1981; Hanrahan, 1982; McElvaney, 1983; Neirinckx, 1980). The most developed generator is one based on adsorption of the parent Ge-68 to a stannic oxide bed (Loc'h, 1980), from which the Ga-68 is eluted with dilute hydrochloric acid. Alternate generators have utilized alumina as adsorbent and EDTA to elute the gallium-68, which presents significant problems in conversion of Ga-68-EDTA complex to other species, given the 68-minute half-life of the nuclide. The invention described herein is preferably directed toward generators of the first type that can be eluted with acid or salt solutions, rather than with chelates such as EDTA.
Given the availability of Ga-68 generators over many years, it is very surprising that no Ga-68-labeled targeting agents have been developed past the point of research article material, and toward routine clinical use. It is one object of the present invention to overcome the radiolabeling problems that have prevented routine clinical preparation of Ga-68-labeled targeting agents.
Gallium is an amphoteric element, which is to say that it displays both basic and acidic reactive properties, and this considerably complicates manipulation of radiogallium. In addition, in dilute solution gallium tends to form non- or poorly-chelated chemical species. The short-lived Ga-68 eluted carrier-free from a generator is present in extremely dilute solution, typically under one picomole (10−12 moles) per milliCurie. It can therefore be particularly prone to the formation of gallates and other species (Hnatowich, 1975; Kulprathipanja and Hnatowich, 1977). This is particularly so as the pH is raised and hydroxy or aqua-ions tend to replace chloride ions in the immediate vicinity of the gallium ions.
Ge-68/Ga-68 generators of the stannous oxide type are usually eluted with a 10–12 mL portion of ultra-pure 1 N hydrochloric acid, providing the Ga-68 daughter in highly dilute form and in the presence of a large amount of hydrochloric acid. Without a purification step, there is also the possibility of eluting other extraneous metal ions along with the Ga-68, and each of these, even in nanomolar amounts, would be typically in 100–10,000 molar excess to the Ga-68. Anionic stannates, can also be eluted which can also complicate carrier-free radiolabeling methods. Once the Ga-68 is obtained, there is then a challenge to bind it to a targeting species, in light of all the above potential problems, and this has been approached in several distinct ways.
First, some workers decided that the carrier-free Ga-68 needed to be mixed with cold gallium to prevent problems seen at high dilution, and so cold gallium was added to Ga-68 eluate prior to admixing with the material to be Ga-68 radiolabeled (Schuhmacher, 1995; Klivényi, 1998). This is cumbersome, and also precludes the preparation of high specific activity Ga-68-labeled species, since the cold added gallium must also be bound by any chelate added during labeling.
A second approach relied on the use of the ‘transchelator’ acetylacetone (2,4-pentanedione) in large excess to bind to radiogallium, essentially using it to out-compete the hydroxy/aqua ions present in the aqueous solution (Lee, 1997; Wu 1997). Unfortunately, this approach is not useful clinically since acetylacetone is a neurological toxin, teratogen and possible mutagen.
A third approach described the evaporation of the Ga-68 eluate from the generator to dryness under a flow of inert gas (Sun, 1996). This was done to remove the excess HCl and to allow the reconstitution of the Ga-68 in another medium. One variation of the method also called for the addition of acetylacetone to protect the Ga-68 while the drying process was continuing (Green, 1993; Tsang, 1993).
Finally, a fourth approach recommended addition of extra concentrated HCl to the Ga-68 generator eluate, until the HCl was 6 N in concentration (Kung, 1990). Then, the Ga-68 in concentrated HCl, was extracted with diethyl ether and reduced to dryness under a stream of nitrogen.
The most advanced technology for clinical application and use of Ga-68 was developed over a three year period, and was based on the evaporation of a reduced elution volume of Ga-68 eluate in 1 N HCl (Goodwin, 1994). Prior to evaporation the Ga-68 was eluted from the Ge-68/Ga-68 generator through an AG1X8 ion exchange filter, and then evaporated on a rotary evaporator, prior to being reconstituted in 10 mM HCl.
Use of Ga-68 carries with it the following concerns: 1) The Ga-68 has a half-life of only 68 minutes, and therefore any methodology used should be fast. 2) Danger to technical personnel is high since the Ga-68 nuclide decays with positron emission at 511 keV making the emergent gamma-rays very difficult to block even with thick (>one inch) lead shielding. 3) In a clinical scenario, the Ga-68 must be obtained sterile and pyrogen-free, and this along with 2) above creates a preference for a method in which manipulations are kept to a minimum. 4) Clinical technical staff have limited chemistry expertise, and are under constant time pressure to produce other unrelated agents during a normal day. They cannot be expected to perform intricate manipulations of the above types in order to effect a Ga-68 labeling.
The above summary of work over a 20-year or so period clearly indicates a need in the art for a viable, rapid and simple method for Ga-68 labeling of specific targeting agents. Disadvantages in the previous methods of Ga-68 labeling have prevented routine adoption of the acid-eluted Ge-68/Ge-68 generators in clinical PET, and have subsisted for over 20 years.